Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
INTERPRETATION MODEL FOR THE UV-VIS SPECTRA OF MICROORGANISMS
FIELD OF INVENTION
This invention relates to the identification of microbial bodies, and more
particularly to the quantitative interpretation of the multiwavelength spectra
of
microorganisms and cells.
BACKGROUND OF THE INVENTION
Multiwavelength Uv-vis spectra of microorganisms and cell suspensions contain
quantitative information on their number, size, shape, chemical composition,
and internal
structure. These properties constitute essential information for the
identification and
classification of microorganisms and cells.
Characterization and classification of living organisms is a major objective
in all
branches of the biological sciences. There are many techniques used for the
characterization of microorganisms such as microscopy, electron microscopy and
ultraviolet microscopy, biomedical characteristics, chemical characterization
and the like.
Typical procedures include cell cultures which are time consuming and
relatively
expensive. For example, the specific information about the cell size and shape
contained
in the photomicrograph is quite limited and many cells have to be counted to
obtain
meaningful statistics.
Microorganisms have differences in chemical composition at the molecular
level.
At the macroscopic level they also have differences in size, shape and cell
morphology.
Different molecular cell compositions result in distinct spectroscopic
patterns.
Accordingly, spectroscopy techniques permit the classification and
identification of
microorganisms by their spectra.
Multiwavelength transmission spectra consist of combined absorption and
scattering phenomena resulting from interaction of light with the
microorganisms or cells
typically suspended in a non-absorbing media. The distribution of intensities
as a function
of wavelength depends on the optical properties of the sample. The complexity
of
microorganisms in terms of their chemical composition and internal structure
makes the
interpretation of their spectral signature a difficult task.
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
In the prior art, spectral signatures may be used to identify an unknown
sample.
However, the sample parameters of the unknown must substantially match those
used to
generate the known signature. In non-laboratory environments rapid
identification of
microorganisms cannot presently be achieved.
Accordingly, what is needed in the art is a model for the interpretation of
the
multiwavelength spectra of microorganisms. Such a model could be employed to
interpret
the spectra of microorganisms in non-laboratory settings. Because the model
can be
dynamically adjusted to match the conditions of the sample, the predicted
spectra can be
constructed and the sample swiftly identified.
It is, therefore, to the effective resolution of the aforementioned problems
and
shortcomings of the prior art that the present invention is directed.
However, in view of the prior art in at the time the present invention was
made, it
was not obvious to those of ordinary skill in the pertinent art how the
identified needs
could be fulfilled.
SUMMARY OF THE INVENTION
The present invention comprises a model for the interpretation of the
multiwavelength spectra of microorganisms. The model is based on light
scattering
theory, spectral deconvolution techniques, and on the approximation of the
frequency
dependent optical properties of the basic constituents of living organisms.
The optical
properties as functions of wavelength, and available literature data on size
and chemical
composition of E. Coli cells and B. globigii spores have been used to
establish the
sensitivity of the calculated spectra to the model parameters and to
demonstrate that the
model cari reproduce the features of experimentally measured spectra. The
model is used
to deconvolute measured spectra in terms of critical parameters necessary for
the detection
and identification of cells, such as the size, dry mass, dipicolinic acid and
the nucleotide
concentration. The present invention has been showed to yield meaningful
estimates of
the size, dry mass, dipicolinic acid and the nucleotide concentration for E.
Coli, P.
agglomerayas, B. subtilis spores, and the vegetative cells and spores of B.
globigii from
experimental data. Reliable estimates for cell size, number, chemical
composition and
indication of the characteristics of their internal structure and invention is
applicable to a
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
wide range of cell types found in diverse environments can be provided.
A method for detecting and identifying microorganisms from a multiwavelength
spectra is provided including the steps of obtaining a specimen, resolving a
sample
spectrum for the specimen, calculating the average turbidity of the sample
spectrum,
normalizing the sample spectrum based on average turbidity, and comparing the
sample
spectrum to a library of known spectra to identify the specimen. The average
turbidity is
calculated by:
1 ''~
z - M ~ zoo
wherein M is the number of discrete data points taken, and io; represents the
turbidity measured at the ith wavelength. Preferably, the sample spectrum
excludes
wavelengths absorbed by a buffer solution in which the specimen is suspended.
In the event that the identity of the specimen cannot be satisfactorily
determined by
comparing the sample spectrum to the library of fingerprints a plurality of
cellular
component values from an optical property database for a known microorganism
are
selected. The plurality of component values may include those for cell
chromophores,
cellular structures, or at least one value of each. A model turbidity spectrum
is calculated
based on the sum of the cellular component values and the sum is compared with
the
sample spectrum to determine whether the sample spectrum is indicative of the
known
microorganism.
Previously, interpretation was limited to particle size and count in the range
of 400
to 820 nm. The present invention now models cellular component values that
contribute to
the spectrum in wavelengths less than 400 nm such as nucleic acid
(approximately at '260
nm) and dipicolinic acid (approximately at 275 nm). Accordingly, modeling of
cellular
components is substantially achieved through a 180 to 1100 nm spectrum range.
As a
plurality of component values may be employed in the model, the number of
quantitative
markers is increased, and thus, the accuracy and precision of the
microorganism
identification.
The model turbidity spectrum is calculated by:
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
M
z~~'o > - Np'~ 4 ~ x= ,~ Q~t,a (m; (~o )~ D)DZ.f (D)dD
where D is the effective particle diameter, Q~t (m (~.o ), D) corresponds to
the Mie
extinction coefficient, m(~,o ) is the complex refractive index, and Np is the
number of
particles per unit volume.
Preferably, the interpretation model divides the predetermined cell's
structure into
values for macrostructure, internal structure, and chromophores wherein the
sum of the
macrostructure, internal structure and chromophores values approximate the
predetermined cell's turbidity spectrum. The equation for the total turbidity
written in
terms of the three distinct populations is:
C.7t;1 ~
~Qext +
i~miOo)~D)DZWD)~D
, Macro
.b
S tructure
~~~'0~ xz ~~ext ~
- Np'e tOZy'o):D)DZWD)~D +
4 , rateraar
Structure
Cxs ~
~Qext
OmtUo)~D)DZ
f
(D)dD
, Other
.
Structure
Where na, (i =1-3) is the refractive index and x; (i = 1-3) is the number
fraction for
each population,
3
~x1 =1
f=I
The volume fraction of each population, vt within the microorganism is given
by:
c~
v; -
Ctotal
Assuming volume addivity, the total concentration, ctorar can be readily
calculated
in terms of the concentration of each population, ct:
N
Ctota! - ~ Ci
i=1
4
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
Where N is the number of populations.
The real and imaginary parts of the complex refractive indices are functions
of the
chemical composition and can be calculated as a weighted sum of the
contributions from
the chromophores within each population:
M
ni -~~lu
M
k; _ ~ Cvikri
Where cul; is mass fraction of the jth chromophore contained in the l~h
population, n;
and k~ real and imaginary refractive indexes of each population, and M is the
total number
of chromophoric groups. Adding the scattering contributions represented in
Paragraph
00023 closes the total mass balance for each ohromophoric group. Substituting
the
relations of Paragraphs 32-33 in the complex refractive index given by:
Substituting the relations of Paragraphs 32-33 in the complex refractive
index,
results the complex refractive index of the ith population:
M M
~1~ jn~ t ~l~ijkij
In i = +
n0 n0
One application that may employ the present invention is that of a biosensor
for
detecting and identifying microorganisms from a multiwavelength spectra. The
biosensor
includes a spectrometer means adapted to measure the spectrum of a fluid
sample. A
processing means is communicatively coupled to the spectrometer means and a
database is
communicatively coupled to the processing means. A plurality of stored
cellular
component values representative of a microorganism model is stored in the
database. The
stored cellular component values may include scattering components such as the
body of
the cell, the cell wall, ribosomes, nucleic acid, cell inclusions and the
like. The stored
cellular component values may also include chromophoric groups such as nucleic
acids,
amino acids, proteins, pigments, and toxins. It should be noted that
chromophoric groups
such as nucleic acids and dipicolinic acid, can be considered to be part of a
particular
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
scattering element (i.e., nuclei, chromosomes), or consider as scattering
elements by
themselves (i.e., plasmids). A plurality of stored cellular component values
are selected
for the model and their sum is calculated to predict the expected turbidity
spectrum. A
signal output means is provided whereby a value for the at least one
quantifiable parameter
of the fluid sample is measured by the spectrometer means and passed to the
processing
means, the processing means accesses the database for a stored quantifiable
parameter in
correlation with the at least one quantifiable parameter, responsive to a
positive
correlation, a signal is generated by the signal output means. A storage means
communicatively coupled to the processing means monitors changes in
quantifiable
parameters in the fluid sample over time.
Accordingly, a first embodiment of the present invention is a method for
detecting
and identifying microorganisms from a multxwavelength spectra including the
steps of
obtaining a sample, acquiring the spectra from the sample, calculating the
average
turbidity of the sample spectra, normalizing the sample spectrum based on the
average
turbidity, and comparing the sample spectrum to a library of known spectra to
identify the
specimen. The average turbidity is calculated by:
I M
Z - ~ ~ Z of
i=I
wherein M is the number of discrete data point wavelengths taken, and io;
represents the
turbidity measured at the ith wavelength and the sample spectrum excludes
wavelengths
absorbed by the suspending medium in which the sample is suspended. The
multiwavelength spectra may utilize polarized light, non-polarized light, or a
combination
thereof.
Another embodiment of the invention includes the steps of obtaining a sample,
acquiring the spectra from the sample, selecting a plurality of component
values from an
optical property database, calculating the turbidity spectrum based on the sum
of the
component values, and comparing the calculated turbidity spectrum with the
sample
spectrum to determine whether the sample spectrum is indicative of a known
microorganism. The spectrum range is between about 180-1100 nm. The plurality
of cellular components values includes those for chromophores and cellular
structures. At
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
least one value of the plurality of cellular component values is a chromophore
and at least
one value of the plurality of cellular component values is a cellular
structure. The model
turbidity spectrum is calculated according to the sum of its macrostructure,
chemical
composition and internal structures and the plurality of component values
include at least
one chromophoric group ( i.e., nucleic acids, chromophoric aminoacids). As in
the first
embodiment, the multiwavelength spectra may include polarized light, non-
polarized light,
or a combination thereof.
The step of selecting the plurality of component values from the optical
property
database includes establishing an interpretation model associated with at
least one
microorganism and selecting the plurality of component values according to the
model.
This is done by identifying an array of cellular elements likely to absorb and
scatter based
on known microbiology of the at least one microorganism, validating the array
of cellular
elements against quantitative data, and associating the validated array with
the at least one
microorganism as the interpretation model.
Alternatively, the model may be derived by selecting a first combination of
cellular
elements from the optical property database, calculating the sum of
contributions from the
first combination to produce a calculated spectrum, comparing the calculated
spectrum
with a reference spectrum of a known microorganism, and iterating through
additional
combinations of cellular elements until the calculated and reference spectrums
correlate.
It is therefore an object of the present invention to provide a method of
identifying
microorganisms that is reliable, efficient and fast.
It is another object of the present invention to provide a biosensor employing
known spectroscopic technology capable of identifying microorganisms
substantially
without manual intervention.
It is to be understood that both the foregoing general description and the
following
detailed description are explanatory and are not restrictive of the invention
as claimed.
The accompanying drawings, which are incorporated in and constitute part of
the
specification, illustrate embodiments of the present invention and together
with the general
description, serve to explain principles of the present invention.
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
These and other important objects, advantages, and features of the invention
will
become clear as this description proceeds.
The invention accordingly comprises the features of construction, combination
of
elements, and arrangement of parts that will be exemplified in the description
set forth
hereinafter and the scope of the invention will be indicated in the claims.
BRIEF DESCRIPTION OF THE DRAWll~TGS
For a fuller understanding of the nature and objects of the invention,
reference
should be made to the following detailed description, taken in connection with
the
accompanying drawings, in which:
FIG. 1 is a diagrammatic of a prokaryotic cell.
FIG. 2 is a schematic representation of the absorption and scattering
components for the quantitative interpretation model.
FIG. 3 is diagrammatic view of an embodiment of the invention utilizing
normalization.
FIG. 4 is diagrammatic view of an embodiment of the invention utilizing
deconvolution.
FIG. 5 is diagrammatic view of an embodiment of the invention utilizing
deconvolution for both polarized and non-polarized light.
FIG. 6 is a diagrammatic view of an embodiment of the invention wherein
calculation models are derived iteratively.
FIG. 7 is a diagrammatic view of an embodiment of the invention wherein
predetermined models define the set of cellular elements selected from the
optical property
database.
FIG. ~ is a diagrammatic view of an embodiment of the invention wherein
calculation models are derived from known microbiology of microorganisms.
FIG. 9 is a normalized optical density spectra from suspension of E. Coli,
8
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
P. agglomerans, and the vegetative cells of B. globigii.
FIG. 10 is a comparison between the normalized optical density spectra
from suspensions of spores and vegetative cells of B. globigii.
FIG. 11 is a comparison between the normalized optical density spectra
from suspension of spores of B. globigii and B. subtilis.
FIG. 12 is a comparison between measured and calculated optical density
spectra of E. coli.
FIG. 13 is a comparison between measured and calculated optical density
spectra ofP. agglorizerans.
FIG. 14 is a comparison between measured and calculated optical density
spectra of B. globigii vegetative cells.
FIG. 15 is a comparison between measured and calculated optical density
spectra of B. globigii spores.
FIG. 16 is a comparison between measured and calculated optical density
spectra of B. subtilis spores.
DETAILED DESCRTPTION OF THE PREFER1ZED EMBODIMENT
The present invention is based on Mie theory, where the volume of the
microorganisms is expressed in terms of an equivalent sphere, and where the
complex
structure of microorganisms is approximated by dividing it into M groups or
populations,
each of which is characterized by its corresponding scattering and absorption
components.
The total scattering and absorption components of the spectrum will be given
by the
weighted sum of the contributions from the selected M structures or
populations:
EQUATION 1:
z(~ ) = N ~ ~ ~ x~ ,~ Q~~., (jn; Uo )~ D)DZ.f (D)dD
o p C4~ t
1=I
9
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
Where xl(i = 1 ~ M) is the number fraction corresponding to each population
such
that,
EQUATION 2
M
x; = I
The complex refractive index m;(~,o) is function of the chemical composition
and
can be calculated as a weighted sum of the contributions from the N
Chromophores within
each population:
EQUATION 3
n n
~~i>~?i i~~likl
ma~~o)_' 1
Tao ~o
Where ~J is mass fraction of the jth chromophore contained in the i~h
population, fz~
and kt correspond to the real and imaginary refractive indices of each
population. Adding
the scattering contributions represented by Equations 1 and 2 closes the total
mass balance
for each chromophoric group. The diameter in Equation 1 can be calculated from
the
closest geometrical approximation to the shape of the microorganisms. Assuming
volume
additivity, the total concentration can be readily calculated in term of the
concentration of
each population or structure.
Prokaryotic cells were used to validate the utility of the present invention.
Prokaryotic cells are unicellular organisms with several architectural regions
as shown in
Fig. 1. appendages (proteins attached to the cell surface) in the form of
flagella and pili
(extensions of the membrane that allow bacteria to stick to substances, and to
each other);
a cell envelope consisting of a capsule (which protects the cell from the
immune system of
its host); cell wall and plasma membrane; and a cytoplasmic region that
contains the cell
genome (total DNA content), ribosomes, and various sorts of inclusions. The
main
scattering elements that can be readily identified are the body of the
microorganism, the
surrounding cell wall, the ribosomes, the nucleic acid structures, and the
inclusions. The
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
absorption components include nucleic acids, nucleotides, and the amino acids
present in
proteins. For some microorganisms, specific pigments and/or toxins may be
present.
A starting point for the modeling of the spectra of prokaryotic cells is to
represent
the complex structure of the microorganisms by dividing it into two main
absorption and
scattering groups or populations (M=2) containing a maximum of three groups of
substances (N=3). The two populations are: (1) the body of the microorganism
and its
characteristic dimensions; and (2) an average representation of the internal
structure
through a characteristic dimension. The chemical composition of these
structures will
consist, as a first approximation, of nucleic acids, dipicolinic acid, and non-
chromophoric
substances such as proteins, lipids, and the like. Fig. 2 shows a schematic
representation of
the model dividing the macrostructure from the internal structure.
An additional simplification results from the low sensitivity shown by the
simulated spectra to the variances of these two populations, and from the
electron
microscopy observations suggesting that the samples of vegetative cells and
spores
analyzed were narrowly distributed. Under these approximations, Equation 1
reduces to:
EQUATION 4:
z(oo)=NP~~4~(xu~~(mr(~o)~y)Di ~'(1"xi)~z~r(nza(~o)~Dz)Dz~
Where the subscript 1 corresponds to the characteristics of the
macrostructure, and
2 to corresponding elements of the internal structure. Equations 3 and 4
constitute the
initial equations for the interpretation of the spectra.
E. Coli (JM 109ATCC #53323) and B, globigii spores (ATCC #9372) were
obtained from the American Type Culture Collection (ATCC) Manassas, Virginia,
P.
agglomera~s and B. subtilis were provided by Los Alamos National Laboratory,
Los
Alamos, New Mexico. Free carboxyl and amino groups (i.e., tyrosine) used for
the
representation of terminal amino acid groups and the n-acetyl/ethyl ester
derivatives used
to represent chromophores imbedded in protein molecules were purchased from
Sigma
Aldrich, Saint Louis Missouri. The dipicolinic acid was also from Sigma-
Aldrich.
11
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
The Uv-vis transmission spectra from the cells suspensions were recorded using
a
diode array spectrometer (HP X443 Hewlett-Packard, Palo Alto, CA) having an
acceptance
angle smaller than 2°. All measurements were conducted at room
temperature using a 1 cm
pathlength cuvette. Prior to running each sample, the spectrometer was zeroed
to account
for any stray light. To avoid the effect of inhomogeneities in the suspending
medium, the
background spectrum was taken using the corresponding suspending media from
the batch
utilized in the preparation of the original sample (sterilized de-ionized
water). To eliminate
concentration and particle number effects, the transmission spectra were
normalized with
the average optical density between 230-900 nm. To illustrate better the
composition
information, the first derivative of the spectra was numerically evaluated.
Because biologically pure cultures do not necessarily imply spectroscopically
pure
materials, special care is taken in washing and separating the cultures. This
is particularly
critical when the growth media has strong absorption characteristics. For
these cases, prior
to the spectroscopy measurements, the cells were washed in sterilized de-
ionized water
according to the following protocol; The microfuge tubes containing the sample
were
placed a Beckman Microfuge 12 and spun for four minutes. The tubes were
removed from
the microfuge and using a 1.0 ml pipette, the supernatant was slowly drawn off
and
discarded. A small amount of the fluid was left in each tube to avoid
disturbing the pellets.
The remaining pellets were resuspended in sterilized deionized water and
vortexed fox few
seconds. The washing process was repeated at least two times. Spectroscopy
measurements were taken to ensure the elimination of the growth media. The
washing
process was repeated until no changes in the spectra were observed. After the
last washing,
the pellet of clean cells was resuspended in sterilized deionized water, which
was also used
to dilute the samples. The level of dilution was selected to yield optical
density values
below 1.2 Absorbance Units (Au). When samples of pure cells (spores and
vegetative
cells) were available, they were directly suspended and diluted in sterilized
de-ionized
water prior to spectroscopy measurements.
The Mie scattering coefficients in Equation 4 were calculated with a computer
program, which includes multiwavelength spectral calculations and has been
adapted to
calculate distributions of particle sizes. This program has been validated
against published
12
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
computer codes and tables. The refractive index of water ho(7,,0) in Equation
2 as a function
of wavelength was calculated from the correlation reported by Thorma,hlen.
The average refractive index pertaining to the scattering groups approximating
the cell
macrostructure were estimated using a two parameter Cauchy equation. The
macrostructure parameters obtained for the microorganisms studied are given in
Table I
together with refractive index values reported in the literature.
TABLE I:
Parameter Literature
Values Values
E. ColiP. B. B. B. VegetativSpores
agglomeglobigiiglobigiisubtilisa cells
cans Veg. spores
Cell
ao 1.3776 1.4538 1.3925 1.49931.4928 -
bo 3034.911984.922849.2 4393.94983.4 - -
3 0 2
ns42 1.3879 1.4605 1.4021 1.51421.5098 1.386- 1.52-
1.400 0.01
77.54 47.59 71.63 24.92 26.75 72-78 18-26
~
water
I
With the exception of P. agglomerates, the estimated refractive indices are
within the
expected range. The reason for this discrepancy is not known at the present
time, but it
translates into lower water content than expected for vegetative cells (see
last row in Table
I).
TABLE II:
13
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
Parameter
estimates
MicroorganismE. B. globigiiB. globigiiP. B. subtilis
Coli veg. spores agglomef~aspores
cells
ns
Volume, um3 1.26 1.230.7820.43 0.43 0.59
Equiv. diameter1.34 1.33 1.14 0.93 1.04
+/- 0.05 ~.m
DNA+RNA 7.70x 3 .64 1. 52 2.02 x 16.84 x
x 10- x 10-I 10-14 10-
~
content/cell 10-14 14 16
+/-
10%
DPA, g/cell - - 4.86 70.06 x10-
+J- 5% x10-14
16
Fraction of 28.5 30.04 41.47 31.17 61.42
internal
structure,
+/- 3%
Average size 80.76 72.84 132 66.98 128
internal structure,
+/- 5 nm
The refractive index parameters for the internal structure were estimated
under the
assumption that primarily non-absorbing proteins compose the internal
scattering
elements. Analysis of the data reported in the literature resulted in the
following estimates:
EQUATION 5:
n(~,o)=1.55+ 5~~0
0
14
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
The optical properties corresponding to chromophoric groups incorporated to
the
scattering elements in Equations 3-4 were estimated. Whenever possible the
measured data
was compared with literature reports and found to be in good agreement.
Application of the interpretation model to the measured spectra results in
quantitative differences between vegetative cells, vegetative cells and
spores, and between
different types of spores. For this purpose equations 4-5 have been
implemented within a
standard Marquardt-Levenberg Least-Squares algorithm. The parameters estimated
include the average size of the microorganism (macrostructure), the average
size of the
internal scattering structures, the volume fraction of the internal structure,
and the
chemical composition in terms of the total nucleotide and dipicolinic acid
concentrations.
Although within the formulation of the interpretation model it is possible to
consider the
effect of the chromophores within each of the absorption and scattering
structures
(Equations 1-4), initially, only two hypothesis have been evaluated, namely
whether the
chromophoric groups absorb and scatter with the macrostructure or as part of
the internal
structure of the microorganisms. The parameter estimation and the evaluation
of the two
hypotheses were conducted conditional upon the values of the refractive index
of the
macrostructure for each microorganism (Table n, and the average values of the
refractive
index of the internal structure (Equation 5). The spectral data used for the
estimation of the
parameters was limited to the range 220-900 nm. This was done to minimize the
effects of
fluctuations in the composition of the suspending media.
There are many issues related to the spectroscopy of microoxganisms and the
identification of their characteristic fingerprints. The spectrometers and the
measurement
of the spectra do not represent a major technical challenge. However, the
sampling
process, the sample preparation, the state of growth of the microorganisms,
and their
natural variability in terms of size, shape and chemical composition can
introduce
considerable differences in their spectral response. For the identification of
characteristic
fingerprint of microorganisms, and for the development of interpretation
models it is
necessary to fix some of these variables. The reproducibility of the spectral
measurements
reported herein has been confined to cell suspensions prepared in sterilized
de-ionized
water.
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
Previous reports have shown that the Uv-vis spectra of different types of
microorganisms may vary considerably. In this report bacteria, vegetative
cells and spores
are analyzed to explore the potential of multiwavelength transmission
measurements to
discriminate between them. Fig. 9 shows the normalized optical density spectra
and the
first derivative spectra (inset) measured for E. Coli, P. agglomef~a~s, and
The vegetative
cells of B. globigii. Notice that although the spectra appear to be somewhat
similar, they
are not identical; this is probably due to differences in chemical
composition, in particular
DNA and RNA. The spectral differences can be appreciated particularly in the
first
derivative spectra, which enhance the differences in chemical composition
(inset Fig. 10).
The similarity in the spectra is expected since these organisms have similar
sizes and water
content. Fig. 11 shows a comparison between the spores and vegetative cells of
B. globigii.
There is a marked difference between the spectra of the spores and the
vegetative cells.
These differences are expected since only the spores contain dipicolinic acid.
Furthermore,
the presence of high concentrations of calcium makes the spores highly
refractive and as a
result their scattering properties are affected. Comparison of the spectra
from B. globigii,
and B. subtilis spores show considerable differences (Fig. 12). The first
derivative spectra
shown in the inset has the same absorption bands, but clearly with varying
intensity. The
experimental measurements of the multiwavelength transmission spectra show
evidence of
quantifiable differences between the bacterial cells and the spores analyzed.
In Fig. 3, the transmission spectra from a sample is acquired 20. Preferably,
the
wavelengths absorbed by the suspending media are excluded. The average
turbidity is
calculated 30 by:
I M
Z ~ M ~ Zoi
i=1
wherein M is the number of discrete data point wavelengths taken, and ioj
represents the
turbidity measured at the i~' wavelength and the sample spectrum excludes
wavelengths
absorbed by the suspending medium in which the sample is suspended. The
spectrum is
normalized 40 based on the mean turbidity 30 excluding the range absorbed by
the
suspending media. A spectra fingerprint database 60 is accessed and the
acquired spectra
20 is compared to known spectra fingerprints in the database 60 for a match.
16
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
Fig. 4 illustrates an embodiment of the invention using deconvolution to
identify the acquired transmission spectra 20 wherein a plurality of cellular
elements are
selected 70 from an optical property database 80 of cellular element
contributions. The
sum of the contributions is calculated 90 from the selected absorptive andlor
scattering
elements selected. An appropriate scattering theory 100 may be applied such as
Mie as
known by those skilled in the art. The calculated spectrum 90 and the observed
spectrum
20 are then compared 110 to determine the presence of the microorganism in the
sample.
In Fig. 5, an alternative embodiment of Fig. 4 is presented wherein non-
polarized 81 and
polarized 82 values are provided to enable more precise deconvolution of the
acquired
spectra 20.
In Fig. 6, a plurality of cellular elements are selected 70 from the optical
property database 80 and the sum of contributions are calculated 90. The
calculated and
observed spectrums are compared 110 and evaluated 120 to determine whether a
match is
achieved under predetermined threshold tolerances. It is preferred that the
spectrums be
compared 'using a least-squares approach, but alternative methods known to
those skilled
in the art are acceptable. If an identification is achieved 130, the
microorganism
associated with the preselected model is deemed to be present in the sample.
Alternatively, if the match is not achieved within threshold tolerates, a new
combination of
selected elements may be executed 140 iteratively and the process repeated.
Fig. 7 illustrates the process of employing preexisting models for
microorganisms in the present invention wherein at least one suspected
microorganism is
established 150 for identification. An array of established sets of cellular
elements is
accessed 170 containing predetermined models for at least one microorganism.
The
established sets 170 are derived from the optical property database 80. A set
of cellular
elements is selected 160 which corresponds to the predetermined model for the
suspected
microorganism previously selected 150 and the sum of contributions is
calculated 90 to
determine if the suspected microorganism is present in a sample.
In Fig. 8, at least one suspected microorganism is established 150 for
identification. The cellular elements likely to absorb andlor scatter are
established 180
based on known microbiology for the at least one suspected microorganism. The
combination of cellular elements selected are then validated 190 against
quantitative data.
17
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
Upon validation, the combination forms a model 170 for the at least one
microorganism.
If validation is not achieved, a new combination of cellular elements are
selected and the
validation process is repeated.
Figs. 13-16 show typical comparisons between measured and calculated spectra
for three vegetative cells and for B. globigii, and B. subtilis spores. The
contribution to the
calculated total optical density from each of the model components, the total
absorption of
the corresponding chromophoric groups calculated as Beer-Lambert absorption,
and the
residuals are also included in the figures. Notice the spectral features
contributed by each
component and the dramatic effect of the chromophoric groups to the total
optical density.
Extinction coefficient is a spectroscopic term applied to a molecular group
for determining
absorption at a particular wavelength. The two structures and the chromophoric
groups
contribute differently over different portions of the spectra. Although the
residuals show
some correlation, it is evident that the present invention adequately
represents the
measured spectra of a large variety of prokaryotic micraorganismS.
The values for the parameters estimated for all the microorganisms studied are
reported in Table II together with their approximate estimations errors. In
addition to the
parameter estimates, Table II also shows the literature data available for the
volume of the
microorganisms and the chromophore concentrations. The estimates of the
particle
volumes and concentrations are within the range of values reported in the
literature that
were obtained with completely different methods. The good agreement with
literature
values is surprising given the simplifying assumptions of the model. There are
two
outstanding values: the first one is the relatively small volume estimated for
P.
agglomerayis, which could explain the low water content reported in Table I.
The second
value corresponds to the low DPA concentration estimate for B. subtilis.
Notice however,
that the DNA concentration estimates are in good agreement with the literature
reports for
this microorganism. The total nucleotide concentration estimated is in
excellent agreement
with the literature for E. Coli. Similarly, the DPA concentration for B.
globigii obtained
from the spectral deconvolution is in good agreement with the values reported
in the
literature for spores.
The correlation observed in the residuals can be explained on the basis of the
approximations and simplifications used. In particular, a spherical equivalent
scattering
18
CA 02494182 2005-02-04
WO 2004/015136 PCT/US2003/024728
approximation has been used. Only the average sizes for each population of
microorganisms and for the internal structure have been estimated. Also, an
average
representation of the total nucleotide absorption has been utilized, and the
contribution of
chromophoric amino acids has been neglected. The last two may account for the
correlation in the residuals apparent in the region between 230-300 nm. The
present
invention may also include hypochromic effects, which are known to alter the
spectral
features of strong chromophoric groups such as DNA and RNA. This novel model
is
capable of representing the spectroscopy behavior of complex biological
structures such as
vegetative cells and spores, and as such, it provides quantitative information
for analysis
and differentiation.
It will be seen that the objects set forth above, and those made apparent from
the
foregoing description, are efficiently attained and since certain changes may
be made in
the above construction without departing from the scope of the invention, it
is intended
that all matters contained in the foregoing description or shown in the
accompanying
drawings shall be interpreted as illustrative and not in a limiting sense.
It is also to be understood that the following claims are intended to cover
all of
the generic and specific features of the invention herein described, and all
statements of
the scope of the invention which, as a matter of language, might be said to
fall
therebetween. As microorganisms pervade nearly every environment on Earth,
affect the
health of billions of humans and animals daily, are critical to numerous
commercial
industries, and are of concern as potential military and terrorist weapons,
the scope of
applications for the present invention should not be limited by exemplary uses
described
herein. Now that the invention has been described,
19
